The complement system comprises a number of serum proteins that function in the body's immune response to infection and tissue injury. Activation of complement can occur via three pathways, the classical pathway involving the binding of complement component C1q to antigen-antibody complexes, the lectin pathway involving binding of mannose binding lectins to antigens, and the alternative pathway involving binding of complement component C3b to an activator surface such as cell wall polysaccharides of yeast and bacterial microorganisms. Activation of complement results in the formation of anaphylatoxins (C3a and C5a), membrane attack complexes (C5b-9), and opsonins (C3b and C4b) that amplify inflammation and destroy foreign and necrotic cells.
Complement activation is regulated by a number of plasma and cell associated proteins. Such proteins inactivate specific steps of the classical, lectin, and/or alternative pathway by regulating the activity of C3/C5 convertases or serving as a cofactor for the factor I cleavage of C3b and/or C4b. These proteins are either soluble plasma proteins or membrane proteins (integral or lipid-anchored) expressed on a variety of cell types. These proteins possess many structural similarities.
Decay Accelerating Factor (DAF)
Decay accelerating factor (DAF, CD55) is a membrane-associated regulatory protein that protects self cells from activation of autologous complement on their surfaces. DAF acts by rapidly dissociating C3 and C5 convertases, the central enzymes of the cascade. DAF possesses the most potent decay accelerating activity of the proteins associated with complement regulation, and acts on both the classical pathway (C4b2a and C4b2a3b) and alternative pathway (C3bBb and C3BbC3b) enzymes. DAF, however, does not have cofactor function.
Structural analyses of DAF have shown that, starting from its N-terminus, it is composed of four ˜60 amino acid-long units followed by a heavily O-glycosylated serine (S) and threonine (T) rich stretch, which is, in turn, linked to a posttranslationally-added glycoinositolphospholipid (GPT) anchor. The amino acid sequence of DAF is shown in FIG. 1 A (SEQ. ID NO: 1). The four 60 amino acid long repeating units are termed complement control protein repeats (CCPs) or short consensus repeats (SCRs). CCPI includes amino acids 35-95 of SEQ. ID NO: 1. CCP2 includes amino acids 97-159; CCP3 includes amino acids 162-221 and CCP includes amino acids 224-284 of SEQ. ID NO: 1. They provide for all of DAF's regulatory activity. The heavily O-glycosylated region serves as a cushion which positions the CCPs at an appropriate distance above the surface membrane. The GPI anchor allows DAF to move freely in the plane of the plasma membrane enabling it to inactivate convertase complexes wherever they assemble.
The critical role that DAF plays in inhibiting complement activation is evident both from natural disease and studies in animal models employing Daf knockout mice. In the human disease paroxysmal nocturnal hemoglobinuria (PNH), mutation in the GPI anchor pathway leading to the absence of DAF renders affected blood cells susceptible to heightened C3b uptake and intravascular hemolysis. In the animal disease models employing the Daf knockout, the absence of DAF renders the mice markedly more susceptible to tissue damage in 1) nephrotoxic serum (NTS) induced nephritis, a model of human membranous glomerulonephritis, 2) dextran sodium sulfate (DSS) induced colitis, a model of inflammatory bowel disease, and 3) anti-acetylcholine receptor (anti-AChR) induced myasthenia gravis, a close model of the human autoimmune disorder.
The nucleotide sequence of a cDNA encoding DAF is shown in FIG. 1B (SEQ. ID NO: 2).
Complement Receptor 1 (CR1)
Complement receptor 1 (CR1 or the C3b receptor, CD35) is another potent regulator of complement activation. Unlike DAF which functions intrinsically to protect the cells that express it, CR1 functions extrinsically on targets of complement attack, e.g. pathogens. CR1 is a larger molecule in that, rather than 4 CCPs, it is comprised of 30 CCPs arranged in 4 groups of 7 CCPs termed long homologous repeats (LHRs). The CCPs and LHRs of CR1 are provided in Table I below. The amino acid residue numbers refer to the amino acid sequence provided in FIG. 2 (SEQ. ID NO: 3). Functional analyses have shown that CR1 possesses both decay accelerating activity and cofactor activity for cleavage of C4b and C3b by the serum enzyme, factor I. Early studies showed that among complement regulators, it is the most potent in this latter activity and that it is the only regulator that promotes both initial cleavage of C3b to iC3b and subsequent cleavage of the iC3b intermediate to C3dg, the surface-bound C3b end product.
Structure-function studies of CR1 have shown that its regulatory activity resides primarily in its three N-terminal LHRs, i.e., LHRs A, B, and C. Functional activity within each 7 CCP LHR is contained essentially in each case in the first 3 CCPs. Recent studies have shown that CR1's potent cofactor activity resides in LHRs B and C, while its decay accelerating activity resides in LHR A.
The nucleotide sequence of a cDNA encoding CR1 is shown in FIG. 3 (SEQ. ID NO: 4).
TABLE 1Amino Acid No.Domain1 or 6-46Leader peptide 47-106CCP1, begin LHR-A107-168CCP2169-238CCP3239-300CCP4301-360CCP4361-423CCP5424-496CCP7, end LHR-A497-556CCP8, begin LHR-B557-618CCP9619-688CCP10689-750CCP11751-810CCP12811-873CCP12874-946CCP14, end LHR-B 947-1006CCP15, begin LHR-C1007-1068CCP151069-1138CCP161139-1200CCP171201-1260CCP181261-1323CCP201324-1399CCP21, end LHR-C1400-1459CCP22, begin LHR-D1460-1521CCP231522-1591CCP241592-1653CCP251654-1713CCP261714-1776CCP271777-1851CCP28, and LHR-D1852-1911CCP291912-1972CCP30
Membrane Cofactor Protein (MCP)
MCP (also known as ‘CD46’) is present on the cell surface of a number of cell types including peripheral blood cells (excluding erythrocytes), cells of epithelial, endothelial and fibroblast lineages, trophoblasts and sperm. MCP has four CCPs and a serine/threonine enriched region in which heavy O-linked glycosylation occurs. MCP also has a transmembrane and cytoplasmic domain. The structure of MCP is provided in Table 2 below with reference to the amino acid sequence of MCP provided in FIG. 4A (SEQ. ID NO: 5). MCP works by binding to the C3b and C4b present on the cell surface thereby targeting C3b and C4b for degradation by factor I, a plasma protease, and thereby destroying any subsequent C3 or C4 convertase activity. Thus, MCP is said to have “cofactor activity”. Because MCP is localized on the cell surface, it protects only the cells on which it is present and is therefore said to act in an intrinsic manner. The sequence of a cDNA encoding human MCP has been reported by Lublin et al, J. Exp. Med., (1988) 168:181-194. The nucleotide sequence of a cDNA encoding MCP is shown in FIG. 4B (SEQ ID NO: 6).
TABLE 2Amino AcidDomain 1-34Leader peptide 35-95CCP  96-158CCP 159-224CCP 225-285CCP 286-314STPB-domain: VSTSSTTKPASSASC-domain: GPRPTYKPPVSNP 315-327Undefined segment 328-351Transmembrane domain 352-361Intracytoplasmic anchor 362-377Cytoplasmic tail one:TYLTDETHREVKFTSL 362-384Cytoplasmic tail two:KADGGAEYATYQTKSTTPAEQRC
Effects of Excessive Activation of Complement
Excessive activation of complement causes damage to normal host tissues in a number of conditions. Some diseases in which complement is known to be activated include systemic lupus erythematosus, acute myocardial infarction, burn, sepsis, stroke and the adult respiratory distress syndrome. Accordingly, it is desirable to have soluble agents that can block complement activation. Such agents would be useful for treating the above-mentioned human diseases and a wide range of other diseases (See Table 3 below). The construction of hybrid complement regulatory proteins has been attempted previously, but with mixed results. For example, a hybrid containing CCPs 1-4 of MCP and CCPs 1-4 of DAF was constructed by Iwata, et al (J. Immunol. 1194, 152:3436). While this hybrid had greater activity in the alternative pathway than either MCP or DAF, it had less activity than DAF alone or DAF plus MCP in the classical pathway. Additionally, in tests of reciprocal chimeric complement inhibitors, one chimeric protein retained the activity of is CD59 and DAF components, while its reciprocal retained only the activity of its DAF component (Fodor, et al, J. Immunol., 1995, 155:4135). Therefore, there is a need for a reliable method for constructing hybrid and chimeric complement regulatory proteins. There is also a need for a hybrid complement regulating protein that possesses the decay accelerating activity of DAF and the co factor activity of CR1.
TABLE 3Potential Clinical Targets of Protein of the InventionAlternative PathwayClassical PathwayReperfusion injuryAutoimmune diseasesCerebral infarction (stroke)Systemic lupus erythematosusAcute myocardial infarctionRheumatoid arthritisHypovolemic shockGlomerulonephritisMultiple organ failureHemolytic anemiaCrush injuryMyasthenia gravisIntestinal ischemiaMultiple sclerosisInflammatory disordersVasculitisAdult respiratory distress syndromeInflammatory bowel diseasesThermal injury (burn & frostbite)Bullous diseasesPost-pump syndrome (cardiopulmonaryUrticariabypass & hemodialysis)Paroxysmal nocturnalCrohn's diseaseHemoglobinuriaSickle cell anemiaCryoglobulinemiaPancreatitisInflammatory disordersAdverse drug reactionsSeptic shock & endotoxemiaRadiographic contrast media allergyTransplant rejectionDrug allergyHyperacute allograftIL-2 induced vascular leakage syndromeTransplant rejectionHyperacute xenograft